The invention relates generally to fluorescent labelling techniques, and more particularly, to the use of 4,7-dichlorofluoresceins for detecting multiple target substances in the same sample.
Many diagnostic and analytical techniques require that multiple target substances in the same sample be labelled with distinguishable fluorescent tages, e.g. Lanier et al, J. Immunol., Vol. 132, pgs. 151-156 (1984)(flow cytometry); Gray et al, Chromosoma, Vol. 73, pgs. 9-27 (1979)(flow system karyotyping); Fung et al, U.S. Pat. No. 4,855,225 (DNA sequencing); and Mayrand et al, Applied and Theoretical Electrophoresis, Vol. 3, pgs. 1-11 (1992)(analysis of electrophoretically separated polymerase chain reaction (PCR) products). This requirement is particularly difficult to satisfy in DNA sequence analysis where at least four spectrally resolvable dyes are needed in most automated sequencing approaches.
Presently, there are two basic approaches to DNA sequence determination: the dideoxy chain termination method, e.g. Sanger et al, Proc. Natl. Acad. Sci., Vol. 74, pgs. 5463-5467 (1977); and the chemical degradation method, e.g. Maxam et al, Proc. Natl. Acad. Sci., Vol. 74, pgs. 560-564 (1977). The chain termination method has been improved in several ways, and serves as the basis for all currently available automated DNA sequencing machines, e.g. Sanger et al, J. Mol. Biol., Vol. 143, pgs. 161-178 (1980); Schreier et al, J. Mol. Biol., Vol. 129, pgs. 169-172 (1979); Smith et al, Nucleic Acids Research, Vol. 13, pgs. 2399-2412 (1985); 35Smith et al, Nature, Vol. 321, pgs. 674-679 (1987); Prober et al, Science, Vol. 238, pgs. 336-341 (1987), Section II, Meth. Enzymol., Vol. 155, pgs. 51-334 (1987); Church et al, Science, Vol 240, pgs. 185-188 (1988); and Connell et al, Biotechniques, Vol. 5, pgs. 342-348 (1987).
Both the chain termination and chemical degradation methods require the generation of one or more sets of labeled DNA fragments, each having a common origin and each terminating with a known base. The set or sets of fragments must then be separated by size to obtain sequence information. In both methods, the DNA fragments are separated by high resolution gel electrophoresis. In most automated DNA sequencing machines, fragments having different terminating bases are labeled with different fluorescent dyes, which are attached either to a primer, e.g. Smith et al (1987, cited above), or to the base of a terminal dideoxynucleotide, e.g. Prober et al (cited above). The labeled fragments are combined and loaded onto the same gel column for electrophoretic separation. Base sequence is determined by analyzing the fluorescent signals emitted by the fragments as they pass a stationary detector during the separation process.
Obtaining a set of dyes to label the different fragments is a major difficulty in such DNA sequencing systems. First, it is difficult to find three or more dyes that do not have significantly overlapping emission bands, since the typical emission band halfwidth for organic fluorescent dyes is about 40-80 nanometers (nm) and the width of the visible spectrum is only about 350-400 nm. Second, even when dyes with non-overlapping emission bands are found, the set may still be unsuitable for DNA sequencing if the respective fluorescent efficiencies are too low. For example, Pringle et al, DNA Core Facilities Newsletter, Vol. 1, pgs. 15-21 (1988), present data indicating that increased gel loading cannot compensate low fluorescent efficiencies. Third, when several fluorescent dyes are used concurrently, excitation becomes difficult because the absorption bands of the dyes are often widely separated. The most efficient excitation occurs when each dye is illuminated at the wavelength corresponding to its absorption band maximum. When several dyes are used one is often forced to make a trade off between the sensitivity of the detection system and the increased cost of providing separate excitation sources for each dye. Fourth, when the number of differently sized fragments in a single column of a gel is greater that a few hundred, the physiochemical properties of the dyes and the means by which they are linked to the fragments become critically important. The charge, molecular weight, and conformation of the dyes and linkers must not adversely affect the electrophoretic mobilities of closely sized fragments so that extensive band broadening occurs or so that band positions on the gel become reversed, thereby destroying the correspondence between the order of bands and the order of the bases in the nucleic acid whose sequence is to be determined. Finally, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the fragments. For example, in the chain termination method, the dyes used to label primers and/or the dideoxy chain terminators must not interfer with the activity of the polymerase or reverse transcriptase employed.
Because of the severe constraints only a few sets of fluorescent dyes have been found that can be used in automated DNA sequencing and in other diagnostic and analytical techniques, e.g. Smith et al (1985, cited above); Prober et al (cited above); Hood et al, European patent application 8500960; and Connell et al (cited above).
In view of the above, many analytical and diagnostic techniques, such as DNA sequencing, would be significantly advanced by the availability of new fluorescent dyes (1) which are physiochemically similar to readily available dyes, (2) which permit detection of spacially overlapping target substances, such as closely spaced bands of DNA on a gel, (3) which extend the number of bases that can be determined on a single gel column by current methods of automated DNA sequencing, and (4) which are amenable for use with a wide range of preparative and manipulative techniques.
The invention is directed to a method of concurrently detecting spacially overlapping target substances using 4,7-dichlorofluorescein dyes, and in particular, methods of DNA sequence determination employing 4,7-dichlorofluorescein dyes. The invention also includes 2xe2x80x2,7xe2x80x2-dichloro-5 (and 6-)carboxy-4,7-dichlorofluorescein defined by Formula I. 
wherein:
Axe2x80x2 is hydrogen, fluoro, chloro, a linking functionality, such as isothiocyanate, succinimidyl carboxylate, or phosphoramidite, or a group, such as carboxyl, sulfonyl, or amino, that may be converted to a linking functionality; preferably Axe2x80x2 is a linking functionality or a group that may be converted to a linking functionality;
Xxe2x80x2 is hydrogen, fluoro or chloro, such that whenever Axe2x80x2 is a substituent of the 6 carbon atom Xxe2x80x2 is a substituent of the 5 carbon atom, and whenever Axe2x80x2 is a substituent of the 5 carbon atom Xxe2x80x2 is a substituent of the 6 carbon atom; preferably, Xxe2x80x2 is hydrogen;
Z3 is hydrogen, fluoro, chloro, a linking functionality, such as isothiocyanate, succinimidyl carboxylate, or phosphoramidite, or a group, such as carboxyl, sulfonyl, or methylamino, that that may be converted to a linking functionality; preferably, Z3 is hydrogen or chloro;
Z4 is hydrogen, fluoro, chloro, a linking functionality, such as isothiocyanate, succinimidyl carboxylate, or phosphoramidite, or a group, such as carboxyl, sulfonyl, or methylamino, that may be converted to a linking functionality; preferably, Z4 is hydrogen or chloro;
Bxe2x80x2 is fluoro, chloro, or an acidic anionic group; preferably, Bxe2x80x2 is carboxyl or sulfonyl, and most preferably Bxe2x80x2 is carboxyl;
and wherein at least one of Axe2x80x2, Z3, and Z4 is a linking functionality or a group that may be converted to a linking functionality. Preferably, only one of Axe2x80x2, Z3, and Z4 is a linking functionality or a group that may be converted to a linking functionality.
The invention also includes kits for carrying out the method of the invention. Generally, kits are provided for detecting a plurality of electrophoretically separated classes of DNA fragments. In particular, kits are included for carrying out DNA sequencing wherein at least one class of primer extension product is fluorescently labelled with a 4,7-dichlorofluorescein dye. Such DNA sequencing kits include kits with dye-labelled primers and, as an alternative embodiment, kits with dye-labelled terminators.
Throughout, the Colour Index (Association of Textile Chemists, 2nd Ed., 1971) carbon numbering scheme is used, i.e. primed numbers refer to carbons in the xanthene structure and unprimed numbers refer to carbons in the 9xe2x80x2-phenyl.
The invention is based in part on the discovery that the fluorescent properties of 4,7-chloro-5- (and 6-)carboxyfluorescein and related dyes are highly favorable for use as molecular probes. Their emission band widths are generally 20-30 percent narrower than analogs lacking the 4,7-dichloro derivatives, their emission and absorption maxima are at wavelengths generally about 10-30 nm higher than analogs lacking the 4,7-dichloro derivatives, and their fluorescent efficiencies are high, in some cases being nearly triple those of analogs lackin the 4,7-dichloro derivatives.
As mentioned above, the invention is a based in part on the discovery of a class of fluorescein dyes that have absoption and emission maxima at ususually long wavelengths, narrow emission band widths and other favorable fluorescent properties. In addition, the invention includes the novel fluorescein analogs defined by Formula I as members of this class of dyes. These dyes permit the assembly of novel sets of spectrally resolvable, physiochemically similar dyes particularly useful in automated DNA sequence analysis.
As used herein the term xe2x80x9cspectrally resolvablexe2x80x9d in reference to a set of dyes means that the fluorescent emission bands of the dyes are sufficiently distinct, i.e. sufficiently non-overlapping, that target substances to which the respective dyes are attached, e.g. polynucleotides, can be distinguished on the basis of the fluorescent signal generated by the respective dyes by standard photodetection systems, e.g. employing a system of band pass filters and photomultiplier tubes, or the like, as exemplified by the systems described in U.S. Pat. Nos. 4,230,558, 4,811,218, or the like, or in Wheeless et al, pgs. 21-76, in Flow Cytometry; Instrumentation and Data Analysis (Academic Press, New York, 1985).
The term xe2x80x9clower alkyxe2x80x9d as used herein directly or in connection with ethers denotes straight-chain and/or branched chain alkyl groups containing from 1-6 carbon atoms, e.g. the term includes methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, and the like. More preferably, the term xe2x80x9clower alkylxe2x80x9d denotes an alkyl having from 1 to 3 carbon atoms.
The term xe2x80x9chaloxe2x80x9d as used herein denotes the halogen atoms fluorine, chlorine, bromine, and iodine; more preferably, the term denotes fluorine or chlorine; and most preferably, the term denotes chlorine.
Preferably, the 4,7-dichloro-5- (and 6-) carboxyfluorescein dyes of the invention include those define by Formula II. 
wherein:
Axe2x80x2, Bxe2x80x2 and Xxe2x80x2 are defined as above;
Z1 is hydrogen or, when taken with Z2, benzo;
Z2, when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, or a linking functionality, or a group, such as carboxyl, sulfonyl, or methylamino, that may be converted to an active linking functionality or when taken with Z1, is benzo;
Z3 and Z4 are separately hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group, that may be converted to a linking functionality;
Z5, when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group that may be converted to a linking functionality, or when taken with Z6, is benzo;
Z6 is hydrogen or, when taken with Z5, is benzo; preferably, when taken alone, Z6 is hydrogen, methyl, ethyl, fluoro, chloro, methoxy, or ethoxy;
and wherein at least one of A, Z2, Z3, Z4, and Z5 is a group that may be converted to an linking functionality. Preferably, only one of A, Z2, Z3, Z4, and Z5 is a group that may be converted to an active linking functionality.
Many dyes for use in the invention are commercially available or can be synthesized by techniques known in the art, e.g. Ghatak et al, J. Ind. Chem. Soc., Vol. 6, pgs. 465-471 (1929); and Khanna et al, U.S. Pat. No. 4,439,358. Alternatively, fluorescein analogs, i.e. A=B=carboxyl, can be synthesized by reacting substituted resorcinol with substituted benzophenone or with substituted trimellitic acid in the presence of propionic acid, as illustrated in the examples. Sulfonylfluoresceins, i.e. A or B is sulfonyl, are synthesized following the methods disclosed by Lee et al, Cytometry, Vol. 10, pgs. 151-164 (1989), modified by substituting appropriate reactants to give 5- or 6-carboxyl- or sulfonylfluorescein products. Preferably, when labelling polynucleotides in DNA sequencing the 5- and 6-isomers of the dyes are used separately because they typically have slightly different electrophoretic mobilities that can lead to band broadening if mixtures of the isomers are used. The 5- and 6-isomers of the dyes are readily separated by reverse phase HPLC, e.g. Edmundson et al, Mol. Immunol., Vol. 21, pg. 561 (1984). Generally, it is believed that the first eluting peak is the 6-isomer and the second eluting peak is the 5-isomer.
Dyes of the invention can be attached to target substances by a variety of means well known in the art. For example, Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, 1989) provides guidance and examples of means for linking dyes to target substances. Substituent A is converted to a linking functionality that can be reacted with a complementary functionality on a target substance to form a linking group. The following table lists illustrative linking functionalities that can be formed whenever A is carboxyl, sulfonyl or amine, suitable complementary functionalities, and the resulting linking groups suitable for use with the invention.
Preferably the linking functionality is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinylamine, or succinimidyl carboxylate whenever the complementary functionality is amine. And preferably the linking functionality is maleimide, or iodoacetamide whenever the complementary functionality is sulfhydryl. Succinimidyl carboxylates can be formed by condensing the 5- and/or 6-carboxyls of the above dyes with N-hydroxysuccinimide using dicyclohexylcarbodiimide (DCC), e.g. as illustrated in examples 6 and 8 of Khanna et al, U.S. Pat. No. 4,318,846, and Kasai et al, Anal. Chem., Vol. 47, pgs. 34-37 (1975). Accordingly, these references are incorporated by reference. Dye phosphoramidites are formed as taught by Stein et al, Gene, Vol. 72, pgs. 333-341 (1988); Fung et al, U.S. Pat. No. 4,757,141; European patent application 89116946.8 filed Sep. 13, 1989; and European patent application 88307934.5 filed Aug. 26, 1988. Substitutents R1, R2, and R3 can take a variety of forms, e.g. as taught by Beaucage et al, Tetrahedron, Vol. 48, pgs. 2223-2311 (1992); Caruthers, pgs. 47-94 in Narang, editor, Synthesis and Applications of DNA and RNA (Academic Press, New York, 1987); and the like. Preferably, R1 and R2, taken separately, are methyl, ethyl, or isopropyl, and R1 and R2, taken together with the nitrogen to which they are attached, is a heterocycle having from four to eight carbon atoms and one to two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. More preferably, R1 and R2, taken together with the nitrogen to which they are attached is morpholino. Preferably, R3 is selected from the group consisting of methyl, chlorophenyl, xcex2-cyanoethyl, methylsulfonylethyl, and nitrophenylethyl. Preferably, the phosphoramidite-derived linking group is oxidized to form a phosphorus(V) linkage, e.g. as taught by Beaucage et al (cited above); Stec et al, PCT application PCT/US91/01010; Beaucage et al, U.S. Pat. No. 5,003,097; or the like.
When dyes of the invention are used to label dideoxynucleotides for DNA sequencing, preferably they are linked to the 5 carbon of pyrimidine bases and to the 7 carbon of 7-deazapurine bases. For example, several suitable base labeling procedures have been reported that can be used with the invention, e.g. Gibson et al, Nucleic Acids Research, Vol. 15, pgs. 6455-6467 (1987); Gebeyehu et al, Nucleic Acids Research, Vol. 15, pgs. 4513-4535 (1987); Haralambidis et al, Nucleic Acids Research, Vol. 15, pgs. 4856-4876 (1987); and the like. Preferably, the linking group between the dye and a base is formed by reacting an N-hydroxysuccinimide (NHS) ester of a dye of the invention with an alkynylamino-derivatized base of a dideoxynucleotide. Preferably, the linking group is 3-carboxyamino-1-propynyl. The synthesis of such alkynylamino-derivatized dideoxynucleotides is taught by Hobbs et al in European patent application No. 87305844.0 and U.S. Pat. No. 5,047,519, which are incorporated herein by reference. Briefly, the alkynylamino-derivatized dideoxynucleotides are formed by placing the appropriate halodideoxynucleoside (usually 5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides as taught by Hobbs et al (cited above)) and Cu(I) in a flask, flushing with Ar to remove air, adding dry DMF, followed by addition of an alkynylamine, triethylamine and Pd(0). The reaction mixture can be stirred for several hours, or until thin layer chromatography indicates consumption of the halodideoxynucleoside. When an unprotected alkynylamine is used, the alkynylamino-nucleoside can be isolated by concetrating the reaction mixture and chromatrographing on silica gel using an eluting solvent which contains ammonium hydroxide to neutralize the hydrohalide generated in the coupling reaction. When a protected alkynylamine is used, methanol/methylene chloride can be added to the reaction mixture, followed by the bicarbonate form of a strongly basic anion exchange resin. The slurry can then be stirred for about 45 minutes, filtered, and the resin rinsed with additional methanol/methylene chloride. The combined filtrates can be concentrated and purified by flash-chromatography on silica gel using a methanol-methylene chloride gradient. The triphosphates are obtained by standard techniques.
Target substances of the invention can be virtually anything that the dyes of the invention can be attached to. Preferably the dyes are covalently attached to the target substances. Target substances include proteins, polypeptides, peptides, polysaccharides, polynucleotides, lipids, and combinations and assemblages thereof, such as chromosomes, nuclei, living cells, such as bacteria, other microorganisms, and mammalian cells, tissues, and the like. As used herein the term xe2x80x9cpolynucleotidexe2x80x9d means a single stranded or double stranded chain of DNA or RNA in the size range of a few bases in length to several thousand bases in length, e.g. from 6 to a few tens to several hundreds or to several thousands of bases in length (if single stranded), or in the size range of a few basepairs in length to several thousand basepairs in length, e.g. from 6 to a few lens to several hundred or to several thousand basepairs in length (if double stranded).
A number of complementary functionalities can be attached to the 5xe2x80x2 or 3xe2x80x2 ends of synthetic oligonucleotides and polynucleotides, e.g. amino groups, Fung et al, U.S. Pat. No. 4,757,141 and Miyoshi et al, U.S. Pat. No. 4,605,735; or sulfhydryl groups, Connolly, Nucleic Acids Research, Vol. 13, pgs. 4485-4502 (1985), and Spoat et al, Nucleic Acids Research, Vol. 15, pgs. 4837-4848 (1987).
Dyes of the invention are particularly well suited for identifying classes of polynucleotides that have been subjected to a biochemical separation procedure, such as gel electrophoresis, where a series of bands or spots of target substances having similar physiochemical properties, e.g. size, conformation, charge, hydrophobicity, or the like, are present in a linear or planar arrangement. As used herein, the term xe2x80x9cbandsxe2x80x9d includes any spacial grouping or aggregation of target substance on the basis of similar or identical physiochemical properties. Usually bands arise in the separation of dye-polynucleotide conjugates by electrophoresis, particularly gel electrophoresis.
Classes of polynucleotides can arise in a variety of contexts. For example, they can arise as products of restriction enzyme digests, or as extension products in polymerase or ligase reactions. Preferably, classes identified in accordance with the invention are defined in terms of terminal nucleotides so that a correspondence is established between the four possible terminal bases and the members of a set of spectrally resolvable dyes. Such sets aare readily assembled from the dyes of the invention by measuring emission and absorption bandwidths with commerically available spectrophotometers. More preferably, the classes arise in the context of the chemical or chain termination methods of DNA sequencing, and most preferably the classes arise in the context of the chain termination method. In either method dye-polynucleotide conjugates are separated by standard gel electrophoretic procedures, e.g. Gould and Matthews, cited above; Rickwood and Hames, Eds., Gel Electrophoresis of Nucleic Acids; A Practical Approach, (IRL Press Limited, London, 1981); or Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1 (Springer-Verlag, Berlin, 1984). Preferably the type of gel is polyacrylamide having a concentration (weight to volume) of between about 2-20 percent. More preferably, the polyacrylamide gel concentration is between about 4-8 percent. Preferably the gel includes a strand separating, or denaturing, agent. Detailed procedures for constructing such gels are given by Maniatis et al., xe2x80x9cFractionation of Low Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98% Formamide or 7 M Urea,xe2x80x9d in Methods in Enzymology, Vol. 65, pgs. 299-305 (1980); Maniatis et al., xe2x80x9cChain Length Determination of Small Double- and Single-Stranded DNA Molecules by Polyacrylamide Gel Electrophoresis,xe2x80x9d Biochemistry, Vol. 14, pgs. 3787-3794, (1975); and Maniatis et al., Molecular Cloning; A Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1982), pgs. 179-185. Accordingly these references are incorporated by reference. The optimal gel concentration, pH, temperature, concentration of denaturing agent, etc. employed in a particular separation depends on many factors, including the size range of the nucleic acids to be separated, their base compositions, whether they are single stranded or double stranded, and the nature of the classes for which information is sought by electrophoresis. Accordingly application of the invention may require standard preliminary testing to optimize conditions for particular separations. By way of example, polynucleotides having sizes in the range of between about 20-300 bases have been separated and detected in accordance with the invention in the following gel: 6 percent polyacrylamide made from 19 parts to 1 part acrylamide to bis-acrylamide, formed in a Tris-borate EDTA buffer at pH 8.3 (measured at 25xc2x0 C.) with 48 percent (weight/volume) urea. The gel was run at 50xc2x0 C.
The dye-polynucleotide conjugates on the gel are illuminated by standard means, e.g. high intensity mercury vapor lamps, lasers, or the like. Preferably, the dye-polynucleotides on the gel are illuminated by laser light generated by a argon ion laser, particularly the 488 and 514 nm emission lines of an agron ion laser. Several argon ion lasers are available commercially which lase simultaneously at these lines, e.g. Cyonics, Ltd. (Sunnyvale, Calif.) Model 2001, or the like.
In the chain termination method, dyes of the invention can be attached to either primers or dideoxynucleotides. Dyes can be linked to a complementary functionality on the 5xe2x80x2 end of the primer, e.g. following the teaching in Fung et al, U.S. Pat. No. 4,757,141 which is incorporated herein by reference; on the base of a primer, e.g. following the teachings of Ward et al, U.S. Pat. No. 4,711,955; directly to the 5xe2x80x2-hydroxyl via a phosphoramidite linking functionality; or on the base of a dideoxynucleotide, e.g. via the alkynylamino linking groups disclosed by Hobbs et al, European patent application No. 87305844.0 which is incorporated herein by reference.
Kits of the invention can take a variety of forms, but usually provide the means for the fluoresecent detection of multiple DNAs separated by size. Kits may be used for detecting amplified nucleic acids separated by size (e.g. by electrophoresis), for DNA sequencing, and the like. Generally, the kits will include either an oligonucleotide labelled with a 4,7-dichlorofluorescein dye, or in an embodiment of the DNA sequencing kit a dye-terminator mix wherein at least one of the dye-terminators is labelled with a 4,7-dichlorofluorecein dye. Usually, the dye-terminator is a dideoxynucleoside triphosphate, as described above, labelled with a fluorescent dye.
Kits for detecting amplified nucleic acids comprise at least one oligonucleotide labelled with a 4,7-dichlorofluorescein dye, an enzyme selected from the group consisting of nucleic acid polymerase and nucleic acid ligase, and a reaction buffer. Whenever the kit includes a DNA polymerase, it further includes a nucleoside triphosphate mix, e.g. a 50 nM aqueous solution of EDTA containing the appropriate concentration of nucleoside triphosphates for a particular application, e.g. amplification, sequencing, or the like. When the kit provides a nucleoside triphosphate mix for DNA sequencing it is understood that such triphosphates include analogs, such as nucleoside-5xe2x80x2-O-(1-thiotriphosphates, e.g. as taught by Lee et al, Nucleic Acids Research, Vol. 20, pgs. 2471-2483 (1992). Nucleic acid polymerases include DNA polymerases, RNA polymerases, and reverse transcriptases, and the like. Preferably, whenever the kit is for PCR amplification, the nucleic acid polymerase is Taq polymerase, e.g. as disclosed by Gelfand, U.S. Pat. No. 4,889,818. Guidance for selecting a PCR reaction buffers and nucleoside triphosphate mixes for particular embodiments can be found in Innis et al, Editors, PCR Protocols: A Guide to Methods and Applications (Academic Press, New York, 1990). A typical 10X PCR reaction buffer comprises 15 mM MgCl2, 500 mM KC, and Tris-HCl, pH 8.3.
Preferably, whenever the kit permits a ligase-based amplification reaction, e.g. as disclosed by Landegren et al, U.S. Pat. No. 4,988,617 or the like, the nucleic acid ligase is a thermostable ligase, such as disclosed by Barany, Proc. Natl. Acad. Sci., Vol. 88, pgs. 189-193 (1991). Guidance for selecting a ligase-based reaction buffer can be found in Landegren et al (cited above), Wu et al, Genomics, Vol. 4, pgs. 560-569 (1989); Barany (cited above), and Nickerson et al, Proc. Natl. Acad. Sci., Vol. 87, pgs. 8923-8927 (1990). A typical ligation reaction buffer comprises 20 mM Tris-HCl, pH 7.6; 50 mM KC; 10 mM MgCl2; 1 mM EDTA; 10 mM NAD+, and 10 mM dithiothreitol.
The dye-labelled oligonucleotides of the kit can have a wide range of lengths, but preferably their length are in the range of 6 to 60 nucleotides. More preferably, the oligonucleotides for ligation kits are in the range of 6 to 30 nucleotides in length, and most preferably, the oligonucleotides for ligation kits are in the range of 16 to 25 nucleotides in length. The particular nucleotide sequence of the oligonucleotides are, of course, dictated by the target sequences sought to be amplified. In embodiments for PCR amplification, selection of oligonucleotides for use as PCR primers is well known in the art, e.g. Innis et al (cited above), Hillier and Green, PCR Methods and Applications, Vol. 1, pgs. 124-128 (1991), and the like.
Preferably, in kits for DNA sequencing wherein dye-terminators are provided, each dideoxynucleoside triphosphate is separately labelled with a dye selected from the set comprising 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2xe2x80x2,7xe2x80x2-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2xe2x80x2,7xe2x80x2-dimethoxy-4xe2x80x2,5xe2x80x2-dichloro-5- and 6-carboxyfluorescein, 2xe2x80x2,7xe2x80x2-dimethoxy-4xe2x80x2,5xe2x80x2-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1xe2x80x2,2xe2x80x2,7xe2x80x2,8xe2x80x2-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1xe2x80x2,2xe2x80x2,7xe2x80x2,8xe2x80x2-dibenzo-4xe2x80x2,5xe2x80x2-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2xe2x80x2,7xe2x80x2-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2xe2x80x2,4xe2x80x2,5xe2x80x2,7xe2x80x2-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein. More preferably, dideoxythymidine triphosphate is labelled with 6-carboxyfluorescein (xe2x80x9c6-FAMxe2x80x9d), dideoxycytidine triphosphate is labelled with 2xe2x80x2,4xe2x80x2,5xe2x80x2,7xe2x80x2-tetrachloro-5-carboxyfluorescein (xe2x80x9c5-ZOExe2x80x9d), dideoxyadenosine triphosphate is labelled with 2xe2x80x2,4xe2x80x2,5xe2x80x2,7xe2x80x2-tetrachloro-4,7-dichloro-5-carboxyfluorescein (xe2x80x9c5-HEXxe2x80x9d), and dideoxyguanosine triphosphate is labelled with 1xe2x80x2,2xe2x80x2,7xe2x80x2,8xe2x80x2-dibenzo-4,7-dichloro-5-carboxyfluorescein (xe2x80x9c5-NANxe2x80x9d). It is understood that dideoxyadenosine includes 2xe2x80x23xe2x80x2-dideoxy-7-deazaasdenosine and dideoxyguanosine includes 2xe2x80x2,3xe2x80x2-dideoxy-7-deazaguanosine and 2xe2x80x2,3xe2x80x2-dideoxy-7-deazainosine, and dideoxythymidine includes 2xe2x80x2,3xe2x80x2-dideoxyuridine. Usually, the dideoxynucleoside triphosphates are labelled by way of a linking group. Preferably, the linking group links a 5 carbon of the 2xe2x80x2,3xe2x80x2-dideoxycytidine or 2xe2x80x2,3xe2x80x2-dideoxyuridine to a 5 or 6 carbon of a dye, and the linking group links a 7 carbon of the 2xe2x80x2,3xe2x80x2-dideoxy-7-deazaadenosine or 2xe2x80x2,3xe2x80x2-dideoxy-7-guanosine or 2xe2x80x2,3xe2x80x2-dideoxy-7-deazainosine to a 5 or 6 carbon of a dye. Preferably, the linking group is carboxyaminoalkynyl, and most preferably, the linking group is 3-carboxyamino-1-propynyl.
Preferably, in kits for DNA sequencing wherein dye-terminators are provided, the nucleic acid polymerase is Sequenase(trademark).